WO2002060925A1

WO2002060925A1 - Method of producing latent antithrombin iii

Info

Publication number: WO2002060925A1
Application number: PCT/US2002/002082
Authority: WO
Inventors: Kimberly Bishop; Michael L. Hayes; Julie S. Pollock; Scott Van Patten
Original assignee: Genzymes Corporation
Priority date: 2001-01-30
Filing date: 2002-01-25
Publication date: 2002-08-08

Abstract

The present invention is directed to a method of producing latent antithrombin III comprising: preparing a solution comprising native ATIII and a buffer, wherin sad buffer contains at least one citrate and at least one glycerol; and heating the solution. The step of healing converts the native ATIII into latent ATIII. The methods of the invention result in a more complete conversion of native ATII to the latent form. The invention also prevents the formation of impurities such as ATIII polymers and oxidation products.

Description

METHOD OF PRODUCING LATENT ANTITHROMBIN III

The present invention relates to novel methods of producing a latent form of antithrombin III (ATIII). More particularly, the present invention relates to a method of producing a latent form of ATIII without the formation of polymers. Additionally, the present invention relates to a method of producing a latent form of ATIII with an increased level of conversion from the native form to the latent form.

ATIII, a member of the serpin family of proteins, is a single chain glycoprotein that functions as an inhibitor of thrombin and other factors in the blood coagulation cascade. ATIII can be found in many organisms which produce the protein in nature, in particular, humans, bovines, and goats. It is synthesized primarily in the liver with a signal peptide of 32 amino acids necessary for its intracellular transport through the endoplasmic reticulum; the peptide is then cleaved prior to secretion. Mourey et al., Biochimiθ 72:599-608 (1990). ATIII is present in serum levels of 12.5 mg/dL.

Decreased levels of ATIII may be found in the serum of individuals who have either a hereditary deficiency of ATIII or an acquired deficiency, which can result from a number of pathologic conditions. It has a molecular weight of approximately 58,000 daltons, containing 432 amino acids and has a carbohydrate content of about 15%. The protein has three disulfide bridges connecting Cys8-128, Cys21- 95 and Cys247-430 and four potential N-linked glycosylation sites located at Asn 96, Asn 135, Asn 155 and Asn 192.

ATIII is also produced transgenically. For example, ATIII currently is produced as recombinant human antithrombin III (r-hATIII) in the milk of transgenic goats. Transgenic r-hATIII is a highly purified, well characterized recombinant glycoprotein. A detailed biochemical comparison of ATIII isolated from the milk of transgenic goats and ATIII isolated from human plasma has been carried out. Transgenic ATIII is comparable to human plasma ATIII with respect to: specific activity, purity, degree of oxidation, amount of aggregates, primary sequence, secondary, and tertiary structure.

The major difference observed between human plasma ATIII and transgenic ATIII is in the glycosylation pattern. Human plasma ATIII is glycosylated with biantennary complex structures that are predominantly disialylated at all four sites. Transgenic ATIII also contains biantennary complex structures at Asn 96, 135 and 192. However, these biantennary oligosaccharides are fucosuylated and are a mix of mono-and disialylated structures. Substitution of N-acetylgalactosamine for galactose is also observed on some of these complex glycans as is substitution of N-glycolyl neuraminic acid for N-acetylneuraminic acid. Human plasma ATIII does not contain either N-glycolyl neuraminic acid or N-acetylgalactosamine on its oligosaccharide chains. Oligomannose and hybrid oligosaccharides are present at Asn 155 of the transgenic ATIII.

This difference in glycosylation may account for the slight difference observed in heparin affinity between human plasma ATIII and transgenic ATIII with transgenic ATIII having a slightly higher overall heparin affinity. The increased heparin affinity of transgenic ATIII does not affect the inhibition of thrombin in the presence of excess heparin.

As used herein, the active native intact form of ATIII is designated the S (stressed) form (S-ATIII). S-ATIII forms a tight binding complex with thrombin (markedly enhanced by the presence of heparin) and other enzymes (not all serpins have heparin affinity). S-ATIII, as well as other serpins, share a common three dimensional structure which can undergo large conformational changes under certain circumstances. (Carrell, R. et al., Structural Mobility of Antithrombin and its Modulation by Heparin, Thromb. Haemost, 78, 516-519 (1997)). Serpins contain a reactive center loop, for example, that is directly involved in the inhibition of their target proteases, which in the case of ATIII includes thrombin. The loop on the serpin is cleaved by the protease, resulting in a conformational change in the serpin that traps the protease, thereby rendering it inactive. This trapping involves the stable insertion of half of the cleaved reactive center loop into a beta sheet within the structure of the serpin and results in a stable protease-serpin complex (e.g., thrombin-ATIII or TAT complexes).

S-ATIII can be cleaved to another native form of ATIII, the relaxed (R)- conformation (R-ATIII), by a variety of enzymes, including thrombin. Evans et al., Biochemistry, 31 :12629-12642 (1992). For example, it has been thought that thrombin binds to a reactive C-terminal loop of ATIII and the resultant complex slowly dissociates releasing thrombin and cleaving off the C-terminal loop of inactive ATIII, resulting in R-ATIII. The cleavage occurs spontaneously, even at cold temperatures. R-ATIII is unable to bind thrombin and has a conformation that is quite different from that of S-ATIII. Certain non-target proteases, such as elastase, can also cleave the loop. However, these proteases do not remain bound to the serpin, resulting in a free, cleaved serpin.

Some serpins, including ATIII, can have the loop inserted into the beta sheet without cleavage by using partial denaturing conditions that destabilize the beta sheet. This form of the protein is called the latent form of ATIII (L-ATIII) and, like the cleaved, native form, is both stable and inactive as a protease inhibitor. Native ATIII may be converted to L-ATIII using mild denaturing conditions which cause a conformational change in the native protein resulting in the insertion of an external loop into a beta sheet in the body of the protein. This treatment leaves the protein intact in terms of amino acid sequence and disulfide bonding.

Surprisingly, an antiangiogenic form of ATIII has been found. It was observed in human tumor cell culture supernatant that was, in fact, shown to be bovine ATIII cleaved on its reactive center loop near the normal thrombin cleavage site. This was presumed to be the result of the culture tumor cells producing an as yet unidentified protease which cleaved bovine ATIII present in the serum used to culture cells. A group of experiments were conducted to more clearly define the nature of this antiangiogenic form of ATIII.

Initially, bovine and human ATIII were purified from plasma and then cleaved on the reactive site loop in vitro using elastase. These preparations were demonstrated to be antiangiogenic using the chick chorioallantoic membrane assay. The elastase-cleaved preparations of ATIII (EC-ATI 11) were also shown to be inhibitors of both bovine capillary endothelial (BCE) cell proliferation and tumor growth in SK-NAS human neuroblastoma model. For example, EC-ATI 11 (relaxed) prepared using recombinant human ATIII (r-hATIII; transgenic) is active in a tumor inhibition assay.

Due to the structural similarity of the L-ATIII to EC-ATI 11 (each with all or part of the reactive center loop inserted into the beta sheet), it was theorized that L- ATIII might also be active in this context. Preparations of ATIII using denaturants that had been shown to result in formation of L-ATIII were made. These preparations proved to inhibit endothelial cell proliferation and tumor growth. Purified preparations of L-ATIII were subsequently made which also were shown to inhibit tumor growth. As a result, administration of L-ATIII as a cancer treatment or as tumor suppressant is currently undergoing clinical trials- It was later shown, however, that these preparations contained primarily polymers of ATIII. Consequently, while L-ATIII has shown promise as a tumor inhibitor there are currently two major problems associated with producing L-ATIII on a large scale: (1) the formation of ATIII polymers during the conversion; and (2) the incomplete conversion from native to latent form.

SUMMARY OF INVENTION

To achieve these and other advantages, and in accordance with the purpose of the invention as embodied and broadly described herein, the present invention, in one aspect, provides a method of preparing latent antithrombin III comprising: preparing a solution comprising native antithrombin III with a buffer, wherein said buffer contains at least one citrate and at least one glycerol; and heating the solution. The step of heating converts the native antithrombin III into latent antithrombin III.

Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates a process for making latent ATIII.

Fig. 2 shows the results of a BCE proliferation assay for cleaved and latent ATIII. Fig. 3 shows the tumor volume for cleaved and latent ATIII.

DESCRIPTION OF THE INVENTION

The invention, in one aspect, provides a method of preparing latent ATIII comprising: preparing a solution comprising native ATIII and a buffer, wherein said buffer contains at least one citrate and at least one glycerol; and heating the solution. The step of heating converts the native ATIII into latent ATIII.

As described above, it is known that mild denaturing conditions such as heating may result in the conversion of the native form of ATIII to the latent form of ATIII. As defined herein the latent form of ATIII, L-ATIII, is ATIII which has been mildly denatured such that it has a heparin affinity and has lost its ability to inhibit thrombin. However, not to be limited as to theory, it was surprisingly discovered that the addition of a buffer containing citrate, such as sodium citrate to a solution comprising the native ATIII resulted in more complete conversion of native ATIII to the L-ATIII upon exposing the solution to mild denaturing conditions. Moreover, the presence of glycerol in the buffer reduced the formation of ATIII polymers, an unexpected property. It is believed that the combination of citrate and glycerol, at optimal concentrations, enhanced the conversion of native ATIII to L-ATIII. The methods of the invention are well suited to both laboratory and large scale production of L-ATIII.

The source material, i.e., native ATIII, for the preparation of L-ATIII may be derived from any organism which produces the protein in nature, in particular, humans, bovines, and goats. ATIII can be isolated from body fluids such as serum, ascites, and urine. ATIII may also be synthesized chemically or biologically, such as by cell culture or recombinant technology, or produced transgenically. Similarly, particular portions and conformations of ATIII that may also be used in the methods of the invention can be isolated from natural sources, produced transgenically, or can be chemically or biologically synthesized, such as by guanidine treatment or in vitro cleavage of ATIII.

Recombinant techniques known in the art include, but are not limited to, gene amplification from DNA using polymerase chain reaction (PCR), gene amplification from RNA using reverse transcriptase PCR and NASBA (nucleic acid sequence based amplifications). In a one embodiment, recombinant L-ATIII, denoted r-L-ATIII, may be prepared from recombinant human antithrombin III (r- hATIII). The r-hATIII may produced by any methods known in the art including, for example, in the milk of transgenic goats.

For use in the methods of the invention, the native ATIII may be purified before addition of the buffer and subsequent conversion to the latent form or after addition of the buffer and/or conversion, i.e., purification of the L-ATIII. Furthermore, it is within the practice of the invention to not purify the native or L- ATIII.

In a one embodiment, the native ATIII is added to a solution with a buffer that contains at least one citrate and at least one glycerol. As described above, the citrate surprisingly aids in the conversion of native ATIII to L-ATIII. In a further embodiment, the citrate is present in the buffer in an amount ranging from, for example, about 10 to about 400 mM, and such as from about 40 to about 300 mM. More preferably, the citrate is present in the buffer in an amount of about 250mM. The range of ATIII to buffer, for example, is from greater than 0 to about 50mg ATIII/mL of buffer, such as from about 5 to about 10 mg/mL. One of skill in the art will be able to determine the amount of buffer necessary depending on the application envisaged. For example, at lower concentrations of buffer, one may get less L-ATIII formation, while at higher concentrations there may be more L-ATIII and/or slower reaction times. Any citrate may be used in the practice of the invention including, but not limited to, sodium citrate and potassium citrate.

Glycerol provides the surprising property of reducing the number of ATIII polymers that are formed during the conversion process. In one embodiment, glycerol for example, may be present in an amount ranging from about 1 to about 40% of the total weight of the buffer, such as from about 10 to about 30% of the total weight of the buffer. In another embodiment, glycerol is present at an amount of about 20% of the total weight of the buffer. One of skill in the art may determine the amount of glycerol desired depending on the application envisaged.

As described above, it is believed that the combination of citrate and glycerol in the buffer solution allowed for more efficient conversion of native ATIII to L-ATIII. These results are reflected in Example 1 , discussed below.

The buffer may also contain sodium chloride, or other salts, acids, and/or bases that may adjust the pH of the buffer and/or solution. In a one embodiment, the pH of the buffer ranges from about 3 to about 7, such as from about 4 to about 6. Not to be limited as to theory, in some cases a lower pH appears to promote faster conversion from the native to latent forms of ATIII.

In one embodiment, native ATIII may be converted to L-ATIII using mild denaturing conditions. This may cause a conformational change in the native protein, resulting in the insertion of an external loop into a beta sheet in the body of the protein. This treatment leaves the protein intact in terms of amino acid sequence and disulfide bonding. Any denaturing process that converts ATIII from the native to the latent form may be within the practice of the invention. Heating, for example, may be used to drive the conversion.

Any temperature that results in conversion from native ATIII to L-ATIII is within the practice of the invention. In a one embodiment, the temperature ranges, for example from about 50°C to about 60°C, however the temperature may be optimized based on the application envisaged. For example, at a temperature above 52°C, one may observe more polymer formation, while at lower temperatures, a slower conversion from the native to the latent forms may be observed. Similarly, the amount of time spent heating will also vary depending on the application envisaged and the temperature chosen. In addition, the temperature may be cycled throughout the preparation process. It is well within the ordinary skill in the art to determine the amount of heating necessary to reach the end products desired. In one embodiment, the solution is heated for about 4 days and in another embodiment for about 6 days.

Prior to heating, one may also add a composition or alter the reaction conditions to reduce oxidation of the ATIII. For example, an overlay of nitrogen may be added to the reaction conditions and/or a methione, such as L-methione may be added to the solution.

In one embodiment, ATIII is diafiltered into the buffer. The subsequent heating allows the ATIII to undergo a controlled conformational change to the lower energy "latent" conformer. The process may be optimized by the addition of a buffer containing a citrate, glycerol, and methionine in order to maximize the amount of ATIII converted to the latent form and minimize the formation of product variants caused by the two identified major routes of degradation: multimer formation (aggregation) and methionine oxidation.

In one embodiment, at least about 50% of the native ATIII is converted to L- ATIII. such as at least about 60% and such as about 80%. In one embodiment, about 97% L-ATIII is produced with 0% native ATIII and less than about 5% polymer.

It is also within the practice of the invention to perform a viral inactivation step before or after conversion to L-ATIII. In one embodiment, the resulting L-ATIII is heated to kill any virus in the product and/or to convert any remaining native ATIII to L-ATIII. In one embodiment, the L-ATIII is heated to about 60°C.

The invention will be illustrated by, but is not intended to be limited to, the following examples.

EXAMPLES

Example 1

Purified r-hATIII (Methyl-Eluate) is concentrated and diafiltered into a buffer, filtered into a sterilized vessel through a sterilizing-grade 0.2 urn membrane filter, and then heated at 50+ 1 °C for 5 days. The stabilizing buffer contains approximately 250 mM sodium citrate, 20% glycerol and 20 mM L-methionine, at a pH 6.0. No polymers were formed and conversion to latent was nearly complete (80%) after about 100 hours.

The purified r-L-ATIII may be concentrated and diafiltered into a formulation buffer to produce the bulk drug substance. A schematic depiction of an example of an r-hATIII purification process and the conversion to r-L-ATIII is shown in Fig. 1. For example, in one embodiment, the source material may be prepared from frozen milk produced by transgenic goats expressing the human ATIII gene. The milk is then thawed, clarified by tangential flow filtration and the ATIII-containing clarified permeate purified for example through a Heparin-column chromatography step. The eluted material is transferred to a dedicated downstream-processing area and is diafiltered to reduce conductivity and processed through an anion-exchange chromatography step. The anion-exchange eluted material is adjusted to high ionic strength with sodium citrate and processed through the final purification step, hydrophobic interaction chromatography. The process results in a highly-purified product meeting specifications for general purity, (SDS-PAGE, and reversed phase HPLC), for immunogenic protein impurities (protein impurity ELISA)and goat DNA.

Example 2

The inventors have optimized the conversion of r-hATIII to both EC-ATI 11 and L-ATIII. Preparations of these two forms have been assayed using the BCE proliferation assay (Fig. 2) and the Lewis lung carcinoma mouse model (Fig. 3). These assays were done with identical replicate samples of a given preparation of each form, and the identity of all samples was blended until final results were obtained. Samples of BSA and untreated r-hATIII were included as negative controls. Based on this data, there is no significant difference between the two forms in terms of their anti-angiogenic or anti-tumor activity.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions and methods of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present description cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.

Claims

WE CLAIM:

1. A method of preparing latent antithrombin III comprising: preparing a solution comprising at least one native antithrombin III and at least one buffer, wherein said at least one buffer contains at least one citrate and at least one glycerol; and heating the solution; wherein the step of heating converts the at least one native antithrombin III into latent antithrombin III.

2. The method according to claim 1 , wherein said at least one native antithrombin III is isolated from at least one organism chosen from humans, bovines, and goats.

3. The method according to claim 1 , wherein said at least one native antithrombin III is isolated from body fluids chosen from serum, ascites, and urine.

4. The method according to claim 1 , wherein said at least one native antithrombin III is produced by chemical synthesis or biological synthesis.

5. The method according to claim 4, wherein said biological synthesis is chosen from cell culture methods, recombinant methods and transgenic methods.

6. The method according to claim 4, wherein said recombinant methods are chosen from gene amplification from DNA using polymerase chain reaction (PCR), gene amplification from RNA using reverse transcriptase PCR and NASBA (nucleic acid sequence based amplifications).

7. The method according to claim 1 , wherein said at least one native antithrombin 111 is purified transgenic antithrombin III.

8. The method according to claim 1 , wherein said native at least one antithrombin III is recombinant antithrombin III.

9. The method according to claim 7, wherein a source for the purified transgenic antithrombin III is milk produced by transgenic goats.

10. The method according to claim 1 , wherein the at least one native antithrombin III is purified before addition of the at least one buffer.

11. The method according to claim 1 , wherein the at least one citrate is sodium citrate.

12. The method according to claim 1 , wherein the at least one citrate is present in an amount ranging from about 10 mM to about 400 mM.

13. The method according to claim 12, wherein the at least one citrate is present in an amount ranging from about 40 mM to about 300 mM.

14. The method according to claim 13, wherein the at least one citrate is present in an amount of about 250 mM.

15. The method according to claim 1 , wherein the at least one glycerol is present in an amount ranging from about 1% to about 40% of the total weight of the buffer.

16. The method according to claim 15, wherein the at least one glycerol is present in an amount ranging from about 10% to about 30% of the total weight of the buffer.

17. The method according to claim 16, wherein the at least one glycerol is present in an amount of about 20% of the total weight of the buffer.

18. The method according to claim 1 , wherein the at least one buffer further contains at least one methionine.

19. The method according to claim 1 , wherein said methionine is L-methionine.

20. The method according to claim 1 , wherein the at least one buffer further contains at least one sodium chloride.

21. The method according to claim 1 , wherein the at least one buffer further contains at least one additional ingredient that may adjust the pH of the at least one buffer and/or the solution chosen from salts, acids and bases.

22. The method according to claim 1 , wherein the pH of the at least one buffer ranges from about 3.0 to about 7.0.

23. The method according to claim 22, wherein the pH of the at least one buffer ranges from about 4.0 to about 6.0.

24. The method according to claim 23, wherein the pH of the at least one buffer is about 6.0.

25. The method according to claim 1 , wherein at least 50% of the at least one native antithrombin III is converted to latent antithrombin III.

26. The method according to claim 1 , wherein at least 60 % of the at least one native antithrombin III is converted to latent antithrombin III.

27. The method according to claim 1 , wherein about 80% of the at least one native antithrombin III is converted to latent antithrombin III.

28. The method according to claim 1 , wherein the ratio of the at least one native antithrombin III to the at least one buffer ranges from about 5 to about 10 mg antithrombin lll/mL of buffer.

29. The method according to claim 28, wherein the ratio of the at least one native antithrombin III to the at least one buffer is about 10 mg antithrombin lll/mL of buffer.

30. The method according to claim 1 , wherein the solution is heated to about 50°C.

31. The method according to claim 1 , wherein the solution is heated to about 50°C for about 4 days.

32. The method according to claim 31 , wherein the solution is heated for about 6 days.

33. The method according to claim 1 , wherein an overlay of nitrogen is be added to the solution.

34. The method according to claim 1 , wherein the at least one native antithrombin III is diafiltered into the at least one buffer.

35. The method according to claim 1 , further comprising the step of viral inactivation of the at least one native antithrombin III before the at least one native antithrombin III is added to the solution.

36. The method according to claim 1 , further comprising the step of heating the latent antithrombin III to a temperature sufficient for killing viruses.